I have encountered a new problem and am at the end of my rope.
I am getting big (both in terms of intensity and in terms of size)
smears of DNA from PCR reactions. It appears to be something odd with
my primers, since a) I get the smear if there is no template present and
b) increasing template decreases the amount and size of the smear DNA,
indicating to me that the legitimate PCR product is in competition with
this smear amplification.
We have tried a variety of things, including increasing temperature
and adding DMSO, glycerol and betaine; none have these have solved the
problem. It has happened with several different batches of primers,
designed to recognize a number of different templates.
I am considering the possibility that the problem may be that I am
dissolving the primers in water, rather than TE, and that as a result I
am getting some depurination that is making a small fraction of the
primers relatively non-specific, causing the formation of a set of
gradually concatenating primer products.
So, my questions are:
1) has anyone else encountered this problem, and if so how did they
fix it?
2) does anyone know whether dissolving primers in water (pH of the
water is 5.6, who knows what the primers do to that) can cause this kind
of problem or can depurinate a small fraction of the primer?
I would appreciate any and all suggestions.
Thanks,
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